Adenoviral Vectors Comprising Partial Deletions of E3

ABSTRACT

This disclosure provides replication-incompetent adenoviral vectors useful in vaccine development and gene therapy. The disclosed vectors comprise a selective deletion of E3 and are particularly useful for preparation of vaccines development and for gene therapy using toxic transgene products that result in vector instability that occurs when the entire E3 domain is deleted.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 17/037,966, filed Sep. 30, 2020, nowallowed, which is a continuation of U.S. patent application Ser. No.16/530,359, filed Aug. 2, 2019, issued as U.S. Pat. No. 10,822,619,which is a continuation of U.S. patent application Ser. No. 15/453,579,filed Mar. 8, 2017, issued as U.S. Pat. No. 10,407,696, which is acontinuation of U.S. patent application Ser. No. 14/190,787, filed Feb.26, 2014, issued as U.S. Pat. No. 9,624,510, which claims priority under35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.61/771,370, filed on Mar. 1, 2013, each of which is hereby incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH Grant Nos. P01AI082282 and U19AI074078, awarded by National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

This disclosure relates generally to adenoviral vectors.

DETAILED DESCRIPTION

This disclosure provides replication-incompetent adenoviral vectorsuseful in vaccine development and gene therapy. These vectors comprise aselective deletion of the viral genomic region E3 but retainanti-apoptotic function otherwise provided by E3-encoded proteins. Thisis achieved either by retaining portions of E3 or by including in thevector open reading frames (ORFs) that encode anti-apoptotic proteins.The disclosed vectors are particularly useful for vaccine developmentand for gene therapy in which the encoded protein products are toxic andresult in vector instability.

In some embodiments, a replication-incompetent adenovirus vectorcomprises a deletion of E3 ORF3, ORF4, ORF5, ORF6, and ORF7; andcomprises at least one open reading frame encoding an anti-apoptoticprotein. The anti-apoptotic protein can be a native protein of theadenovirus, such as the proteins encoded by ORF8 or ORF9, or can be anapoptotic protein from a different source, e.g., a p53 inhibitor,Bcl-XL, BCL2, BCL2L1, BCL2A1, BAG1, TRAF1, BIRC3, BIRC5, BAK1, cIAP1,c-IAP2, XIAP, or API5. These proteins and nucleotide sequences encodingthem are well known in the art. A replication-incompetent adenovirusvector can comprise one open reading frame encoding an anti-apoptoticprotein or can comprise several such open reading frames (e.g., 2, 3,4).

An adenovirus vector can be rendered replication-incompetent by variousmeans, including, but not limited to, a complete deletion of E1 or afunctional deletion of E1 (i.e., a deletion of less than the entire E1aand E1b loci, but sufficient to disable the function of the E1genes, andmutations at functional sites).

The disclosed vectors can be generated using basic cloning techniquesand can be used thereafter to express a variety of different proteinproducts.

In some embodiments, the serotype of the replication-incompetentadenovirus vector is a human serotype (e.g., a serotype of group A,group B, group C, group D, group E, group F). Human serotypes include,but are not limited to, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad11, Ad20, Ad21,Ad22, Ad23, Ad24, Ad25, Ad26, Ad28, Ad34, Ad35, Ad40, Ad41, Ad48, Ad49,and Ad50.

In other embodiments, the serotype of the replication-incompetentadenovirus vector is a chimpanzee serotype (e.g., Ad C1, Ad C3, Ad C6,Ad C7, Ad C68).

Replication-incompetent adenovirus vectors disclosed herein areparticularly useful when an antigenic protein is toxic to cell machineryupon expression by the vector. For example, the disclosedreplication-incompetent adenovirus vectors carrying a coding sequencefor HIVgp140 can be readily rescued, express the gp140 protein, andremain stable after 12 serial passages.

Accordingly, in some embodiments, the antigenic protein is all or anantigenic portion of an HIV-1 envelope protein. In some embodiments, theantigenic protein is all or an antigenic portion of an HCV envelopeprotein. In some embodiments, the antigenic protein is all or anantigenic portion of a protein of M. tuberculosis. In some embodiments,the antigenic protein is all or an antigenic portion of a protein ofPlasmodium (e.g., P. falciparum).

In some embodiments, the antigenic protein is all or an antigenicportion of a protein of an infectious eukaryotic organism, such as aPlasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodiumovale, Plasmodium malariae Plasmodium diarrhea), and fungi such asCandida (e.g., Candida albicans), Aspergillus (e.g., Aspergillusfumigatus), Cryptococcus (e.g., Cryptococcus neoformans), Histoplasma(e.g., Histoplasma capsulatum), Pneumocystis (e.g., Pneumocystisjirovecii), and Coccidioides (e.g., Coccidioides immitis).

In some embodiments, the antigenic protein is all or an antigenicportion of a protein of an infectious virus, such as an influenza virus,retrovirus (e.g., HIV, Rous Sarcoma Virus (RSV), human endogenousretrovirus K (HERV-K)), human endogenous retrovirus K (HERV-K),papillomavirus (e.g., human papilloma virus), picornavirus (e.g.,Hepatitis A, Poliovirus), hepadnavirus (e.g., Hepatitis B), flavivirus(e.g., Hepatitis C, Yellow Fever virus, Dengue Fever virus, Japaneseencephalitis virus, West Nile virus), togavirus (e.g., chikungunyavirus, Eastern equine encephalitis (EEE) virus, Western equineencephalitis (WEE) virus, Venezuelan equine encephalitis (VEE) virus),herpesvirus (e.g., Cytomegalovirus), paramyxovirus (Parainfluenza virus,Pneumonia virus, Bronchiolitis virus, common cold virus, Measles virus,Mumps virus), rhabdovirus (e.g., Rabies virus), Filovirus (e.g., Ebolavirus), bunyavirus (e.g., Hantavirus, Rift Valley Fever virus),calicivirus (e.g., Norovirus), or reovirus (e.g., Rotavirus,Epstein-Barr virus, Herpes simplex virus types 1 & 2).

In some embodiments, the antigenic protein is all or an antigenicportion of a protein of an infectious gram-negative bacterium orgram-positive bacterium, Bacillus (e.g., Bacillus anthracis),Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium Leprae),Shigella (e.g., Shigella sonnei, Shigella dysenteriae, Shigellaflexneri), Helicobacter (e.g., Helicobacter pylori), Salmonella (e.g.,Salmonella enterica, Salmonella typhi, Salmonella typhimurium),Neisseria (e.g., Neisseria gonorrhoeae, Neisseria meningitidis),Moraxella (e.g., Moraxella catarrhalis), Haemophilus (e.g., Haemophilusinfluenzae), Klebsiella (e.g., Klebsiella pneumoniae), Legionella (e.g.,Legionella pneumophila), Pseudomonas (e.g., Pseudomonas aeruginosa),Acinetobacter (e.g., Acinetobacter baumannii), Listeria (e.g., Listeriamonocytogenes), Staphylococcus (e.g., methicillin-resistant,multidrug-resistant, or oxacillin-resistant Staphylococcus aureus),Streptococcus (e.g., Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae), Corynebacterium (e.g., Corynebacteriumdiphtheria), Clostridium (e.g., Clostridium botulinum, Clostridiumtetani, Clostridium difficile), Chlamydia (e.g., Chlamydia pneumonia,Chlamydia trachomatis), Camphylobacter (e.g., Camphylobacter jejuni),Bordetella (e.g., Bordetella pertussis), Enterococcus (e.g.,Enterococcus faecalis, Enterococcus faecum, Vancomycin-resistantenterococcus (VRE)), Vibrio (e.g., Vibrio cholerae), Yersinia (e.g.,Yersinia pestis), Burkholderia (e.g., Burkholderia cepacia complex),Coxiella (e.g., Coxiella burnetti), Francisella (e.g., Francisellatularensis), and Escherichia (e.g., enterotoxigenic, enterohemorrhagicor Shiga toxin-producing E. coli, such as ETEC, EHEC, EPEC, EIEC,EAEC)).

Production, purification and quality control procedures for Ad vectorsare well established.¹⁷ Once a vector backbone is created, molecularcloning can be used to create an adenoviral plasmid comprising a codingsequence for an antigenic protein (“transgene”). The plasmid can betransfected into packaging cells that provide E1 of a suitableadenovirus serotype in trans. Packaging cells are well known in the art,and cells lines such as HEK293 can be used for this purpose. Viralparticles are then harvested once plaques become visible. Fresh cellscan then be infected to ensure continued replication of the adenovirus.Quality can be assessed using Southern blotting or other methods, suchas restriction enzyme mapping, sequences, and PCR, to confirm thepresence of the transgene and the lack of gene rearrangements orundesired deletions.

Vaccine compositions comprising adenovirus particles made usingreplication-incompetent adenovirus vectors disclosed herein can be usedto induce immunity against the encoded antigenic protein.

Vaccines can be formulated using standard techniques and can comprise,in addition to a replication-incompetent adenovirus vector encoding adesired protein, a pharmaceutically acceptable vehicle, such asphosphate-buffered saline (PBS) or other buffers, as well as othercomponents such as antibacterial and antifungal agents, isotonic andabsorption delaying agents, adjuvants, and the like. In some embodimentsvaccine compositions are administered in combination with one or moreother vaccines.

Dosage units of vaccine compositions can be provided. Such dosage unitstypically comprise 10⁸ to 10¹¹ adenoviral particles (e.g., 10⁸, 5×10⁸,10⁹, 5×10⁹, 10¹⁰, 5×10¹⁰, 10¹¹, 10⁸ to 10⁹, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰,10⁹ to 10¹¹, 5×10⁸ to 10⁹, 5×10⁸ to 10¹⁰, 5×10⁹ to 10¹⁰, 5×10⁹ to 10¹¹,10⁸ to 5×10⁹, 10⁸ to 5×10¹⁰, 10⁹ to 5×10¹⁰, 10⁹ to 10¹¹, 5×10⁸ to 5×10⁹,5×10⁸ to 5×10¹⁰, 5×10⁹ to 5×10¹⁰, 5×10⁹ to 10¹¹).

Immune responses against one or more encoded proteins or antigenicportions thereof can be induced using the disclosed vaccine compositionsor dosage units. Methods of administration include, but are not limitedto, mucosal (e.g., intranasal), intraperitoneal, intramuscular,intravenous, and oral administration. Immune responses can be assessedusing suitable methods known in the art, as disclosed, for example, inWO2012/02483.

Those skilled in the art will appreciate that there are numerousvariations and permutations of the above described embodiments that fallwithin the scope of the appended claims.

EXAMPLE 1

Construction of pAdC6 020

The E3 region of AdC6 contains 5052 base pairs (bp), with a total ofnine open reading frames: ORF1 is 325 bp; ORF2 is 624 bp; ORF3 is 531bp; ORF4 is 684 bp; ORF5 is 612 bp; ORF6 is 867 bp; ORF7 is 276 bp; ORF8is 435 bp; and ORF9 is 408 bp. ORF3, ORF4, ORF5, ORF6 and ORF7 weredeleted to create the vector “AdC6 020” (the total length of the deletedsequence was 3218 bp).

EXAMPLE 2

Construction of Stable Adenoviral Vectors Comprising HIV EnvelopeSequences

pAdC6 020 was used to create two vectors encoding HIV envelopesequences, “AdC6 020-HIVgp140-DU172” and “AdC6 020-HIVgp140-DU422.” TheHIV strains that served as donors for the envelope sequences wereisolated from clinical materials. The coding sequences werecodon-optimized to permit enhanced expression in human cells. To createthe pAdC6 020 plasmid, PCR-based cloning strategies were used to obtainthe fragment without ORF3, ORF4, ORF5, ORF6 and ORF7 of E3 region. Byusing that PCR product, the pXY-E3 deleted plasmid was generated. BothpXY-E3 deleted (donor for insert) and pC6 E3 deleted (donor forbackbone) plasmids were digested with PspX I and Nco I restrictionenzymes to result in the pC6 E3 modified plasmid. Finally the modifiedE3 fragment was excised out from pC6 E3 modified plasmid by Sbf Idigestion and cloned into pAdC6 000-eGFP plasmid (E1 deleted only) withE3 removed by Sbf I digestion. This resulted in pAdC6 020-eGFP. AdC6vectors comprising the HIV coding sequences and a deletion of E1 or adeletion of E1 and of all the ORFs of E3 could not be rescued. Similarproblems were encountered using adenovirus vectors of human serotype 5(Ad5) and 26 (Ad26). Upon modifying the E3 deletion within AdC6, Ad5 andAd26, however, all of those vectors could readily be rescued. Thesevectors passed quality control assays including restriction enzymemapping, and Western blots showed that vectors expressed HIV-1 gp140upon transfection of cells. These vectors were stable after 12sequential passages shown by restriction enzyme mapping. Vectors rescuedsuccessfully by modified E3 region are summarized in the followingtable.

Viral Western Blot Viral Vector Name Rescued Stability for transgeneGenome AdC6 020-HIVgp140-DU172 yes stable yes correct AdC6020-HIVgp140-DU422 yes stable yes correct AdC7 010-HIVgp140-DU172 yesstable yes correct AdC7 010-HIVgp140-DU422 yes stable yes correct AdC6020-SIVgp160 yes N.D. N.D. correct AdC6 020-HIVgag yes N.D. N.D. N.D.Ad5 060-HIVgp140-DU422 yes stable yes correct Ad5 060-SIVgp160 yes N.D.N.D. N.D. Ad26 011-HIVgp140-DU422 yes N.D. N.D. correct N.D.—NotDetermined

What is claimed is:
 1. A method of inducing an immune response in a subject, the method comprising administering to the subject a replication-incompetent adenovirus vector comprising: a) open reading frame (ORF) ORF1 and ORF2 of E3 genomic region; b) selective deletion of E3 genomic region consisting of ORF3, ORF4, ORF5, ORF6, and ORF7; and, c) at least one ORF encoding a non-adenoviral anti-apoptotic protein.
 2. The method of claim 1, wherein the anti-apoptotic protein is selected from the group consisting of a p53 inhibitor, Bcl-XL, BCL2, BCL2L1, BCL2A1, BAG1, TRAF1, BIRC3, BIRC5, BAK1, cIAP1, c-IAP2, XIAP, and API5.
 3. The method of claim 1, wherein the replication-incompetent adenovirus vector is a human serotype.
 4. The method of claim 1, wherein the replication-incompetent adenovirus vector has group A serotype, group B serotype, group C serotype, group D serotype, group E serotype, or group F serotype.
 5. The method of claim 1, wherein the replication-incompetent adenovirus vector has serotype Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad11, Ad20, Ad21, Ad22, Ad23, Ad24, Ad25, Ad26, Ad28, Ad34, Ad35, Ad40, Ad41, Ad48, Ad49, or Ad50.
 6. The method of claim 1, wherein the replication-incompetent adenovirus vector has serotype AdC1, AdC3, AdC6, AdC7, or AdC68.
 7. The method of claim 1, wherein the replication-incompetent adenovirus comprises a deletion in E1 genomic region.
 8. The method of claim 1, wherein the replication-incompetent adenovirus vector further comprises an ORF encoding an antigenic protein.
 9. A replication-incompetent adenovirus vector comprising: a) open reading frame (ORF) ORF1 and ORF2 of E3 genomic region; b) selective deletion of E3 genomic region consisting of ORF3, ORF4, ORF5, ORF6, and ORF7; c) at least one ORF selected from the group consisting of ORF8 and ORF9; and d) at least one ORF encoding an antigenic protein.
 10. The replication-incompetent adenovirus vector of claim 9, which is a human serotype.
 11. The replication-incompetent adenovirus vector of claim 9, which has a serotype of a group selected from the group consisting of group A, group B, group C, group D, group E, and group F.
 12. The replication-incompetent adenovirus vector of claim 9, which has a serotype selected from the group consisting of Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad11, Ad20, Ad21, Ad22, Ad23, Ad24, Ad25, Ad26, Ad28, Ad34, Ad35, Ad40, Ad41, Ad48, Ad49, and Ad50.
 13. The replication-incompetent adenovirus vector of claim 9, which has a serotype selected from the group consisting of Ad C1, Ad C3, Ad C6, Ad C7, and Ad C68.
 14. The replication-incompetent adenovirus vector of claim 9, comprising a deletion in E1.
 15. An immunogenic composition comprising the replication-incompetent adenovirus vector of claim
 9. 16. A dosage unit of the immunogenic composition of claim 15, which comprises 108 to 1011 adenoviral particles.
 17. A method of inducing an immune response to the antigenic protein in a subject, comprising administering to the subject the immunogenic composition of claim
 15. 18. A packaging cell line comprising the replication-incompetent adenovirus vector of claim
 9. 19. The packaging cell line of claim 18 comprising HEK293 cells.
 20. The replication-incompetent adenovirus vector of claim 9, which has the serotype Ad C7. 